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Mutagenesis and Genetic Screens General pathway for mutational dissection of a biological process “Forward Genetics” Fig. 12-39 General pathway for mutational dissection of a biological process “Forward Genetics” Fig. 12-39 Classification of Mutant Type Test for type of mutation recovered: haplo-insufficient null Fig. 12-38 Test for type of mutation recovered: haplo-insufficient null Fig. 12-38 Test for type of mutation recovered: haplo-insufficient leaky Fig. 12-38 Test for type of mutation recovered: gain-of-function mutation Fig. 12-38 Test for type of mutation recovered: neomorphic mutation Fig. 12-38 General pathway for mutational dissection of a biological process “Forward Genetics” Fig. 12-39 From phenotype to gene • Once an interesting mutant is found and chromosome characterized, we want to find the gene in which the mutant occurred • Positional cloning – First use genetic mapping – Then use chromosome walking contig candidate genes mutation Candidate-gene approach • If the mutated gene is localized to a sequenced region of the chromosome, then look for genes that could be involved in the process under study • Last step: confirm gene identification – Rescue of phenotype – Mutations in same gene in different alleles Insertional mutagenesis • Alternative to chromosome walking – To reduce time and effort required to identify mutant gene • Insert piece of DNA that disrupts genes – Inserts randomly in chromosomes • Make collection of individuals – Each with insertion in different place • Screen collection for phenotypes • Use inserted DNA to identify mutated gene Insertional mutagens • Transposable elements – Mobile elements jump from introduced DNA • e.g., P elements in Drosophila – Or start with a small number of nonautonomous elements – Mobilize by introducing active element • e.g., AC/DS elements in plants • Single-insertion elements – e.g., T-DNA in plants • Once insert, can’t move again Genome-Wide Phenotypic Analysis: “Phenomics” High-Throughput Genetics Applications of genomics approaches to genetics High-throughput genetic screens • Some genetic screens are relatively straightforward – e.g., For a visible phenotype like eye color • If phenotype is subtle or needs to be measured, the screen is more time consuming – Examples • Seed weight • Behavioral traits Industrial setting for screens 2002 Para digm Genetics, Inc. All rights reserved. Used with permission. High-throughput genetic screen • Paradigm Genetics, Inc. performs “phenotypic profiling” • Take measurements of mutants’ physical and chemical parameters – e.g., plant height, leaf size, root density, and nutrient utilization • Different developmental times: 2002 Para digm Genetics, Inc. All rights reserved. Used with permission. compare to wild type Finding random mutations in your gene of interest (or every gene in the genome) • Random insertion of transposons • Random point mutations/indels Screening an insertion library • PCR used to find insertion • One primer complementary to insert • Other primer complementary to gene • If get an amplification product then you have insertion • Sequence product for exact location PCR primers insert gene Z PCR amplification insert gene Z + – amplification product on gel indicates presence of insert near gene P element piggyBac Summary of P element Gene Disruption Project TILLING • Method for finding mutations produced by chemical mutagens in specific genes • Chemical mutagenesis – Usually produces point mutations – Very high mutagenic efficiency – Generally gives more subtle phenotypes than insertions • e.g., hypomorphs, temperature sensitive mutants TILLING in Arabidopsis I • EMS used to mutagenize Arabidopsis • Grow individual mutagenized lines • Make primers flanking gene of interest • Amplify using PCR EMS mutagenize seed gene Z WT gene Z mutant PCR amplification from wild type and mutant WT mutant TILLING in Arabidopsis II • Denature DNA from pools of mutant lines • Allow to hybridize to wild-type DNA • Detect mismatches in hybridized DNA – Denaturing HPLC – Cel I enzyme cuts at mismatches • Sequence to identify site of mutation ATGCGGACTG |||||| ||| + TACGCCGGAC ATGCGG |||||| TACGCC CTG ||| GAC Cel 1 Arabidopsis TILLING Project Summary I • Forward genetics – Mutation to gene function – Genetic screens – Cloning genes identified in screens • Genomics approaches to forward genetics – High-throughput genetic screens – Insertional mutagenesis – Activation tagging – Enhancer trapping and gene trapping